Translocations Through Natural Membranes

Mitchell, Peter

doi:10.1002/9780470122747.ch2

Cited by 92 publications

(39 citation statements)

References 201 publications

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“…With regard to ion-gradient-driven permeases, the chemiosmotic hypothesis of Peter Mitchell (66)(67)(68) has been supported strongly by the experiments of West (112) and West & Mitchell (113,114) and demonstrated quantitatively in bacterial membrane vesicles (41)(42)(43). Thus, accumulation of a wide variety of solutes against a concentration gradient is driven by an electrochemical H + gradient (Δμ̄H + ) composed of an electrical component (ΔΨ; interior negative) and/or a pH gradient (ΔpH; interior alkaline).…”

Section: Introductionmentioning

confidence: 73%

Lessons From Lactose Permease

Guan

Kaback

2006

Annu. Rev. Biophys. Biomol. Struct.

319

417

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An X-ray structure of the lactose permease of Escherichia coli (LacY) in an inward-facing conformation has been solved. LacY contains N-and C-terminal domains, each with six transmembrane helices, positioned pseudosymmetrically. Ligand is bound at the apex of a hydrophilic cavity in the approximate middle of the molecule. Residues involved in substrate binding and H + translocation are aligned parallel to the membrane at the same level and may be exposed to a water-filled cavity in both the inward-and outward-facing conformations, thereby allowing both sugar and H + release directly into either cavity. These structural features may explain why LacY catalyzes galactoside/H + symport in both directions utilizing the same residues. A working model for the mechanism is presented that involves alternating access of both the sugar-and H + -binding sites to either side of the membrane.

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Section: Introductionmentioning

confidence: 73%

Lessons From Lactose Permease

Guan

Kaback

2006

Annu. Rev. Biophys. Biomol. Struct.

319

417

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“…5A, blue) and occluded (Fig. 5A, gray) (41). In this view, the intermediate conformation is induced by sugar binding and leads to lowering of the activation energy barrier for the transition between the inward-facing and outward-facing conformers.…”

Section: Discussionmentioning

confidence: 99%

Apo-intermediate in the transport cycle of lactose permease (LacY)

Madej¹,

Soro²,

Kaback

2012

Proc. Natl. Acad. Sci. U.S.A.

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The lactose permease (LacY) catalyzes coupled stoichiometric symport of a galactoside and an H + . Crystal structures reveal 12, mostly irregular, transmembrane α-helices surrounding a cavity with sugar-and H + -binding sites at the apex, which is accessible from the cytoplasm and sealed on the periplasmic side (an inward-facing conformer). An outward-facing model has also been proposed based on biochemical and spectroscopic measurements, as well as the X-ray structure of a related symporter. Converging lines of evidence demonstrate that LacY functions by an alternating access mechanism. Here, we generate a model for an apo-intermediate of LacY based on crystallographic coordinates of LacY and the oligopeptide/H + symporter. The model exhibits a conformation with an occluded cavity inaccessible from either side of the membrane. Furthermore, kinetic considerations and double electron-electron resonance measurements suggest that another occluded conformer with bound sugar exists during turnover. An energy profile for symport is also presented. proteins represents the largest family of secondary transporters, with members from Archaea to Homo sapiens (1, 2). MFS proteins catalyze transport of a wide range of substrates, including amines, acids, amino acids, sugars, peptides, and antibiotics, in many instances, by transducing the energy stored in an H + electrochemical gradient into a concentration gradient of substrate. The lactose permease of Escherichia coli (LacY), which catalyzes the coupled transport of a galactopyranoside and an H + (galactoside/H + symport) (3-6), is arguably the most extensively studied member of the MFS (7,8). Essentially all 417 amino acyl side chains in LacY have been mutagenized (9), and functional analyses of the mutants reveals that fewer than 10 side chains play a central role in the symport mechanism ( Fig. 1 A and B): Glu126 (helix IV), Arg144 (helix V), and Trp151 (helix V) are directly involved in galactoside recognition and binding; Tyr236 (helix VII), Glu269 (helix VIII), and His322 (helix X) are involved in both H + translocation and affinity for sugar; and Arg302 (helix IX) and Glu325 (helix X) play important roles in H + translocation (7, 10). In the available inward-facing crystal structures of , these residues are located at the apex of a deep central hydrophilic cavity, which is open to the cytoplasm only ( Fig. 1 A and B). The cavity is formed by 12 mostly irregular transmembrane helices organized in two pseudosymmetrical 6-helix bundles (helices I-VI and helices VII-XII) (11)(12)(13)(14).A six-step kinetic model for lactose/H + symport has been proposed (15, 16) (Fig. 1C). The steps include (i) deprotonation of LacY, (ii) a conformational change allowing the galactoside-and H + -binding sites to become accessible to the other side of the membrane, (iii) protonation of LacY, (iv) substrate binding, (v) a global conformational change in which the protonated LacY with bound sugar returns to the initial inward-facing conformation, and (vi) dissociation of sugar. This seque...

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“…Accordingly to the chemiosmotic concept of Peter Mitchell [11][12][13], an electrochemical H + gradient (Δ̃H+) composed of an electrical (the membrane potential, Δ ) and a chemical (a pH gradient, ΔpH) component drives the steady-state accumulation of a wide variety of substrates …”

Section: Introductionmentioning

confidence: 99%

Function, Structure, and Evolution of the Major Facilitator Superfamily: The LacY Manifesto

Madej

2014

Advances in Biology

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The major facilitator superfamily (MFS) is a diverse group of secondary transporters with members found in all kingdoms of life. A paradigm for MFS is the lactose permease (LacY) of Escherichia coli, which couples the stoichiometric translocation of a galactopyranoside and an + across the cytoplasmic membrane. LacY has been the test bed for the development of many methods applied for the analysis of transport proteins. X-ray structures of an inward-facing conformation and the most recent structure of an almost occluded conformation confirm many conclusions from previous studies. Although structure models are critical, they are insufficient to explain the catalysis of transport. The clues to understanding transport are based on the principles of enzyme kinetics. Secondary transport is a dynamic process-static snapshots of X-ray crystallography describe it only partially. However, without structural information, the underlying chemistry is virtually impossible to conclude. A large body of biochemical/biophysical data derived from systematic studies of site-directed mutants in LacY suggests residues critically involved in the catalysis, and a working model for the symport mechanism that involves alternating access of the binding site is presented. The general concepts derived from the bacterial LacY are examined for their relevance to other MFS transporters.

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Translocations Through Natural Membranes

Cited by 92 publications

References 201 publications

Lessons From Lactose Permease

Lessons From Lactose Permease

Apo-intermediate in the transport cycle of lactose permease (LacY)

Function, Structure, and Evolution of the Major Facilitator Superfamily: The LacY Manifesto

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